RPA-CRISPR reaction detection system for detecting equine arteritis virus, African equine pestivirus and / or Hendea virus as well as kit and application of RPA-CRISPR reaction detection system

Abstract

The invention discloses an RPA-CRISPR reaction detection system for detecting equine arteritis virus, African equine pestivirus and / or Hendea virus as well as a kit and application of the RPA-CRISPR reaction detection system, the reaction detection system comprises an RPA amplification system and a CRISPR-Cas detection system, the RPA amplification system comprises an RPA primer pair, the CRISPR-Cas detection system comprises a crRNA sequence, a probe and an RPA amplification product, the sequences of the RPA primer pair are as shown in SEQ ID NO.1 to SEQ ID NO.6, the sequences of the crRNA are as shown in SEQ ID NO.7 to SEQ ID NO.11, and the sequences of the probe are as shown in SEQ ID NO.25 to SEQ ID NO.27. The invention further discloses a kit for detecting the content of the crRNA in the kit. According to the invention, the reaction buffer solution, the probe concentration, the reaction time and the like in a CRISPR-Cas detection system are optimized, and the detection method has good specificity, sensitivity and repeatability on detection of EAV, AHSV and HeV, so that the RPA-CRISPR / Cas multiple detection method has good application prospects in detection and diagnosis of equine pathogens in laboratories and on site.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to an RPA-CRISPR reaction detection system and a kit and application thereof for detecting equine arteritis virus, African horse sickness virus and / or Hendra virus, specifically an RPA-CRISPR / Cas13a, 13b double detection system, an RPA-CRISPR / Cas13a, 13b, 12a triple detection system, etc. Background Art

[0002] In recent years, the horse breeding industry has grown vigorously, but the emergence of horse pathogens has posed a serious threat to the development of this industry. Equine arteritis virus (EAV) is a single-stranded positive-strand RNA virus. It was first discovered in Ohio, USA, and the virus was isolated from aborted equine fetuses and identified as the Bucyrus strain. It is mainly infected through the respiratory and reproductive tracts, with edema, hemorrhage and abortion in pregnant horses as the main clinical features. It currently exists in many countries in the world and is a global infectious disease of horses. The World Organization for Animal Health (OIE) has classified this disease as a Category II infectious disease, which is a major quarantine disease for horses. The genome of African horse sickness virus (AHSV) is a double-stranded RNA containing 10 segments. It is mainly transmitted by blood-sucking insects, causing fever, edema and internal bleeding, with a mortality rate of over 90%. The World Organization for Animal Health (WOAH) has classified African horse sickness as a legally reportable disease, and my country has classified it as a Category I animal disease. Hendra virus (HeV) is a single-stranded negative-sense RNA virus belonging to the Hennipavirus genus of the Paramyxoviridae family. It is highly pathogenic and a rare zoonotic pathogen. The virus was first discovered in a horse farm in Australia in 1994, causing multiple cases of severe febrile respiratory diseases with obvious respiratory symptoms and high mortality. Hendra virus has become one of the pathogens that must be tested by my country's customs, ports and entry-exit management departments.

[0003] At present, the methods for detecting these three viruses include virus isolation and identification, nucleic acid-based detection methods, and protein-based detection methods. The virus isolation and identification method is the standard method for EAV detection approved by the OIE, and is also the most classic method for AHSV diagnosis. However, this method has a long experimental cycle and high sample requirements, and is not suitable for large-scale detection. Nucleic acid-based detection methods such as real-time fluorescence quantitative reverse transcription PCR and reverse transcription-loop-mediated isothermal amplification (RT-LAMP) have improved sensitivity and shortened time, but have high requirements for instruments and operators, cannot be used for on-site instant detection, and are easily contaminated by aerosols, resulting in false positives. Protein-based detection methods such as virus neutralization tests, complement fixation tests, and enzyme-linked immunosorbent assays also have shortcomings such as cumbersome operation processes and high costs. Therefore, it is particularly important to establish a method with high sensitivity, good specificity, and the ability to quickly, simply, and instantly detect EAV, AHSV, and HeV.

[0004] Recombinase polymerase amplification (RPA) is a new isothermal rapid nucleic acid amplification technology that has been widely used in the detection of bacteria, fungi, viruses and other fields. This technology uses recombinase, single-stranded binding proteins (SSB), DNA polymerase and deoxyribonucleoside triphosphate (dNTP) to replace the chain breaking thermal cycling process of ordinary PCR. It is simple to operate and does not rely on complex instruments and equipment. It can achieve rapid amplification of DNA or RNA at a lower temperature (usually 37°C), and the amplified product can be obtained in 30 minutes.

[0005] The clustered regularly interspaced short palindromic repeats and CRISPR associated proteins (CRISPR-Cas) system originates from the adaptive immune system in prokaryotic cells and has been used as an effective tool for genome editing and nucleic acid diagnosis. It can recognize and bind to the target sequence in a specific manner through single-stranded guide RNA (CRISPR ribonucleic acid, crRNA) to form a crRNA-Cas enzyme-target sequence ternary complex, thereby stimulating the cleavage activity of the Cas enzyme. There are many types of CRISPR-Cas systems, including Cas9, Cas12a, Cas12b, Cas13 and Cas14, among which Cas12 and Cas13 are widely used in bacteria, virus and parasite detection due to their special "side branch cleavage" activity. For example, Gootenberg et al. developed the SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) platform, which detects Zika virus and dengue virus based on the CRISPR / Cas13a system, and has outstanding applications in distinguishing different pathogens and human genotyping; Khan et al. detected SARS-CoV-2 based on the SHERLOCK system, with good specificity and a detection limit as low as 6.75 copies / μL, which was 100% consistent with the RT-PCR test results.

[0006] The detection sensitivity of the CRISPR system alone is low. In order to improve the sensitivity of the CRISPR-Cas detection method, researchers have further explored the combination of nucleic acid amplification methods with CRISPR technology, by amplifying the target sequence to increase the concentration of the targeted nucleic acid sequence. Traditional nucleic acid amplification methods such as polymerase chain amplification have high requirements for instruments and operating techniques, cannot be detected on-site, and have limited applicability. With the development of amplification technology, isothermal amplification methods such as loop-mediated isothermal amplification (LAMP) and RPA have gradually shown their advantages. In recent years, more and more studies have combined RPA with the CRISPR-Cas system, taking advantage of the high efficiency, economy and portability of RPA, especially rapid amplification at a temperature compatible with the CRISPR reaction, thereby significantly improving the sensitivity and specificity of detection, reducing the possibility of off-target, and reducing false positives. For example, Miao et al. coupled RPA with CRISPR / Cas13a to detect Nipah virus, with good specificity and the ability to detect as low as 10 3copies / μL target molecule; Lei et al. combined LAMP and CRISPR / Cas12a systems to establish a porcine circovirus detection method, which was 100% consistent with the quantitative PCR method when evaluating clinical blood samples; Dong et al. combined RPA with CRISPR / EsCas13d targeting to detect Japanese encephalitis virus in real time. In order to be more suitable for pathogen diagnosis in real-life scenarios, researchers have further developed dual or multiple CRISPR / Cas detection platforms to meet the simultaneous detection of multiple targets, promoting the application and development of this technology. For example, Zhang et al. used a dual lateral chromatography detection method based on RPA-CRISPR-Cas12a / 13a to quickly and simultaneously detect HPV16 and HPV18; Tian et al. used Cas12a and Cas13a systems to develop a dual-gene detection technology for SARS-CoV-2 and African swine fever virus, which showed 100% sensitivity and specificity for clinical sample analysis.

[0007] Therefore, in order to better meet the needs of clinical diagnosis, it is extremely important to establish a rapid on-site diagnostic method for equine arteritis virus, African horse sickness virus, and Hendra virus. In view of the emergence of mixed virus infection, it is of great practical significance to further establish a method that can simultaneously detect these three horse pathogens. At present, there are relatively few studies based on CRISPR technology to detect EAV, AHSV, and HeV, and there are no reports on combining RPA with CRISPR-Cas system for the detection of equine arteritis virus, African horse sickness virus, and / or Hendra virus. Summary of the invention

[0008] In view of at least one of the deficiencies in the prior art, the present invention first developed a dual CRISPR-Cas13a / 13b platform to achieve single-tube simultaneous detection of equine arteritis virus (EAV) and African horse sickness virus (AHSV); on this basis, a CRISPR-Cas13a / 13b / 12a triple detection method was further established to achieve single-tube simultaneous detection of EAV, AHSV and Hendra virus (HeV). The principle of detecting three targets based on the triple CRISPR / Cas system is as follows: Figure 1 As shown, after extracting viral nucleic acid from the sample mixture, RPA is used to amplify viral cDNA and transcribe EAV and AHSV templates in vitro, and then the target gene is recognized and cut by the triple CRISPR / Cas system, and finally the fluorescent signal is released for detection. This method can quickly detect EAV, AHSV and HeV individually or simultaneously, providing important application value.

[0009] To achieve the above object, the technical solution adopted by the present invention includes:

[0010] The first aspect of the present invention is to provide an RPA-CRISPR reaction detection system for detecting equine arteritis virus, African horse sickness virus and / or Hendra virus, which includes an RPA amplification system and a CRISPR-Cas detection system; wherein, in the RPA amplification system, the primers for detecting equine arteritis virus are shown in SEQ ID NO.1 to SEQ ID NO.2, the primers for detecting African horse sickness virus are shown in SEQ ID NO.3 to SEQ ID NO.4, and the primers for detecting Hendra virus are shown in SEQ ID NO.5 to SEQ ID NO.6. The above RPA-CRISPR reaction detection system can realize the detection of one virus, two viruses or three viruses.

[0011] Furthermore, in the CRISPR-Cas detection system, Cas13a is used to detect equine arteritis virus, Cas13b is used to detect African horse sickness virus, and Cas12a is used to detect Hendra virus; the CRISPR-Cas detection system includes crRNA sequences, probes, and RPA amplification products.

[0012] Furthermore, due to the characteristic of Cas13 enzyme to cut RNA, a T7 RNA polymerase promoter sequence needs to be attached to the 5′ end of its corresponding RPA primer to allow T7 transcription.

[0013] Further, the crRNA sequence for detecting equine arteritis virus is shown in SEQ ID NO.7, and the probe sequence is shown in SEQ ID NO.25; the crRNA sequence for detecting African horse sickness virus is shown in SEQ ID NO.8 to SEQ ID NO.11, and the probe sequence is shown in SEQ ID NO.26; the crRNA sequence for detecting Hendra virus is shown in SEQ ID NO.12, and the probe sequence is shown in SEQ ID NO.27; the 5' end and 3' end of each of the probes are respectively labeled with different fluorescent groups and quenching groups. The above CRISPR-Cas detection system uses 1 EAV-crRNA, 4 AHSV-crRNAs and / or 1 HeV-crRNA.

[0014] Furthermore, the transcription template sequence of the crRNA sequence for detecting equine arteritis virus is shown as SEQ ID NO.13 to SEQ ID NO.14, the transcription template sequence of the crRNA sequence for detecting African horse sickness virus is shown as SEQ ID NO.15 to SEQ ID NO.22, and the transcription template sequence of the crRNA sequence for detecting Hendra virus is shown as SEQ ID NO.23 to SEQ ID NO.24.

[0015] Specifically, the transcription template sequence of AHSV-crRNA1 of the sequence shown in SEQ ID NO.8 is shown as SEQ ID NO.15 to SEQ ID NO.16; the transcription template sequence of AHSV-crRNA2 of the sequence shown in SEQ ID NO.9 is shown as SEQ ID NO.17 to SEQ ID NO.18; the transcription template sequence of AHSV-crRNA3 of the sequence shown in SEQ ID NO.10 is shown as SEQ ID NO.19 to SEQ ID NO.20; the transcription template sequence of AHSV-crRNA4 of the sequence shown in SEQ ID NO.11 is shown as SEQ ID NO.21 to SEQ ID NO.22.

[0016] Furthermore, the probe includes a ssRNA-FQ (FAM-ssRNA) probe, a ssRNA-RQ (ROX-ssRNA) probe, and a ssDNA-HQ (HEX-ssDNA) probe; wherein the ssRNA-FQ probe is used for Cas13a determination (equine arteritis virus), the ssRNA-RQ probe is used for Cas13b determination (African horse sickness virus), and the ssDNA-HQ probe is used for Cas12a determination (Hendra virus); the 5' end of the ssRNA-FQ probe is labeled with a FAM group, and the 3' end is labeled with a BHQ1 group; the 5' end of the ssRNA-RQ probe is labeled with a ROX group, and the 3' end is labeled with a BHQ2 group; the 5' end of the ssDNA-HQ probe is labeled with a HEX group, and the 3' end is labeled with a BHQ3 group.

[0017] The specific sequence information involved in the above RPA-CRISPR reaction detection system is shown in Table 1 below:

[0018] Table 1 - Sequence information

[0019]

[0020]

[0021]

[0022] Further, the RPA-CRISPR reaction detection system includes an RPA amplification system and a CRISPR-Cas13a / Cas13b dual detection system for detecting equine arteritis virus and African horse sickness virus; wherein, based on the total volume of the reaction system of 25 μL, the CRISPR-Cas13a / Cas13b dual detection system includes 2.5 μL Cas13 reaction buffer (10×), 2 μL EAV-RNA, 2 μL AHSV-RNA, 1 μM of each crRNA, 0.5 μM LwaCas13a, 1 μM PsmCas13b, 100 nM ssRNA-FQ probe, 100 nM ssRNA-RQ probe and nuclease-free water; wherein, LwaCas13a is used to detect equine arteritis virus, and the ssRNA-FQ probe is a probe for detecting equine arteritis virus; PsmCas13b is used to detect African horse sickness virus, and the ssRNA-RQ probe is a probe for detecting African horse sickness virus.

[0023] Furthermore, the detection limit of the CRISPR-Cas13a / Cas13b dual detection system for both viruses was 10 2 copies / μL.

[0024] Further, the RPA-CRISPR reaction detection system includes an RPA amplification system and a CRISPR-Cas13a / Cas13b / Cas12a triple detection system for detecting equine arteritis virus, African horse sickness virus and Hendra virus; wherein, based on the total volume of the reaction system of 25 μL, the CRISPR-Cas13a / Cas13b / Cas12a triple detection system includes 2.5 μL Cas13 reaction buffer (10×), 2 μL EAV-RNA, 2 μL AHSV-RNA, 2 μL HeV-DNA, 1 μM of each crRNA, 0.5 μM LwaCas13a, 1 μM PsmCas13b, 0.5 μM LbaCas12a, 400 nM ssRNA-FQ probe, 600 nM ssRNA-RQ probe, 600 nM ssDNA-HQ probe and nuclease-free water; LwaCas13a is used to detect equine arteritis virus, and the ssRNA-FQ probe is a probe for detecting equine arteritis virus; PsmCas13b is used to detect African horse sickness virus, and the ssRNA-RQ probe is a probe for detecting African horse sickness virus; LbaCas12a is used to detect Hendra virus, and the ssRNA-HQ probe is a probe for detecting Hendra virus.

[0025] Furthermore, the detection limit of the CRISPR-Cas13a / Cas13b / Cas12a triple detection system for the three viruses is 10 3 copies / μL.

[0026] Furthermore, the Cas13 reaction buffer (10×) contains 20 mM HEPES, 50 mM KCl, 5 mM MgCl2, 100 μg / mL BSA, pH 7.0.

[0027] Furthermore, the RPA amplification system includes 29.5 μL reaction buffer, 2.4 μL of each RPA primer, 11.2 μL ddH2O, 2 μL cDNA template, and 2.5 μL MgOAc; wherein the final concentration of each RPA primer in the RPA amplification system is 10 μM. The cDNA template can be prepared by conventional methods or kits, and the RPA amplification system is a single RNA amplification system or a multiple RPA amplification system, preferably a single RNA amplification system, that is, amplifying one virus at a time; wherein, RPA amplification is directly used, and agarose gel electrophoresis is used for detection, and the detection limit of each virus is 10 3 copies / μL.

[0028] It is understandable that the composition and dosage of the above system are all optimal reaction systems. Under the premise of ensuring the amplification effect and detection results, the dosage of each component can be adjusted within an appropriate range.

[0029] The second aspect of the present invention is to provide a kit for detecting equine arteritis virus, African horse sickness virus and / or Hendra virus, which comprises the RPA-CRISPR reaction detection system described in any one of the first aspects.

[0030] Furthermore, the above-mentioned kit may also include reagents for preparing cDNA templates, reagents for preparing crRNA, etc.

[0031] A third aspect of the present invention is to provide a method for detecting equine arteritis virus, African horse sickness virus and / or Hendra virus, comprising the steps of:

[0032] S1. After extracting viral nucleic acid from the sample, use the RPA amplification system to amplify each viral cDNA template, and purify the RPA amplification product; wherein the purified equine arteritis virus amplification product and the African horse sickness virus amplification product are transcribed in vitro into respective RNA templates, and the purified Hendra virus amplification product is a DNA template;

[0033] S2, using the CRISPR-Cas detection system to identify and cut the target gene in the RNA template and / or DNA template obtained in step S2;

[0034] S3, determining the detection result based on the fluorescence value or the visual effect under blue light;

[0035] Wherein, the RPA amplification system and the CRISPR-Cas detection system are as described in any one of the first aspects.

[0036] Furthermore, in step S1, the sample includes a horse nose swab sample.

[0037] Furthermore, in step S1, the reaction conditions of the RPA amplification system are: incubation at 37° C. for 30 minutes.

[0038] Furthermore, in step S2, the reaction conditions of the CRISPR-Cas detection system are: 37° C., isothermal amplification for 10 to 30 min (preferably, the double detection system reacts for 20 min, and the triple detection system reacts for 10 min), and the fluorescence intensity is measured every 30 seconds.

[0039] Furthermore, the above-mentioned viral cDNA templates can be prepared by conventional methods; for example, viral RNA is extracted and reverse transcribed from a sample mixture to obtain a viral cDNA template.

[0040] It is understandable that the above reaction conditions and fluorescence intensity measurement time are all optimal conditions, and the condition values ​​adjusted within an appropriate range are all within the protection scope of the present invention, such as ±20%, ±10%, ±5%, etc.

[0041] Furthermore, in step S3, the detection of the visual effect under the blue light includes: after the reaction is completed, performing visual detection under a blue light device and taking photos to record the results.

[0042] Furthermore, in step S3, if the fluorescence value at the end of the reaction exceeds twice the fluorescence value of the negative control, it is judged as positive, otherwise it is negative.

[0043] Furthermore, when it is an RPA-CRISPR dual detection method, the CRISPR-Cas detection system can detect at least one of equine arteritis virus and African horse sickness virus; when the CRISPR-Cas detection system contains only EAV template, an amplification signal appears in the FAM channel, and green fluorescence is exhibited under blue light irradiation; when the CRISPR-Cas detection system contains only AHSV template, an amplification signal appears in the ROX channel, and red fluorescence is exhibited under blue light irradiation; when two targets exist at the same time, amplification signals appear in both the FAM and ROX channels, and yellow fluorescence is exhibited under blue light irradiation.

[0044] Furthermore, when it is an RPA-CRISPR triple detection method, the CRISPR-Cas detection system can detect at least one of equine arteritis virus, African horse sickness virus, and Hendra virus, specifically: when the CRISPR-Cas detection system contains only EAV, AHSV or HeV templates, only the corresponding single fluorescence channel can collect fluorescence, and other channels cannot detect fluorescence; when the CRISPR-Cas detection system contains EAV and AHSV targets, the FAM and ROX channels produce fluorescence, and other channels cannot detect fluorescence; when the CRISPR-Cas detection system contains EAV and HeV targets, the FAM and HeX channels produce fluorescence, and other channels cannot detect fluorescence; when the CRISPR-Cas detection system contains AHSV and HeV targets, the ROX and HEX channels produce fluorescence, and other channels cannot detect fluorescence; when the three targets exist at the same time, all three channels can detect fluorescence.

[0045] The third aspect of the present invention is to provide an application of the RPA-CRISPR reaction detection system described in any one of the first aspects, or the kit described in any one of the second aspects, which is selected from at least one of the following applications: application in detecting equine arteritis virus, African horse sickness virus and / or Hendra virus for non-diagnostic treatment purposes, and application in screening agents or methods for reducing the concentration of equine arteritis virus, African horse sickness virus and / or Hendra virus.

[0046] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0047] The research of CRISPR-Cas system in the prior art mainly focuses on Cas12a and Cas13a enzymes. Compared with the dual detection method based on Cas13a and Cas13b established by the invention herein, Cas12a needs to rely on the PAM (protospacer adjacent motif, PAM) sequence in the target sequence to exert its cutting characteristics, which is limited to the design of target sequence screening and crRNA, narrows the detection range, reduces the practicality of the system as a comprehensive diagnostic tool, and Cas13a and Cas13b are not dependent on the PAM sequence, which simplifies the crRNA design and broadens its application range; after developing the dual detection method of Cas13a and Cas13b, the present invention further adopts the Cas12a enzyme, thereby realizing the simultaneous detection of three viruses; and compared with the detection method combining the CRISPR system with the microwell array system by Ackerman et al., the method established by the present invention does not need to formulate a specific chip, saves the detection cost, and does not need to pre-treat the crRNA or the detection template, the operation process is simple, and its application range is broadened.

[0048] The present invention first established an RPA-CRISPR / Cas13a, 13b dual detection platform to achieve single-tube dual detection of EAV and AHSV. The method is fast and convenient, can complete the detection within one hour, and can identify the results by naked eye, showing good specificity and repeatability. The sensitivity of RPA-CRISPR / Cas13a, 13b in detecting equine arteritis virus and African horse sickness virus is 10 2 copies / μL, which improves the sensitivity compared with the RPA method alone.

[0049] The present invention further developed a triple detection platform based on RPA-CRISPR / Cas13a, 13b, 12a, which can simultaneously detect three equine pathogens, EAV, AHSV and HeV. The method has high specificity and good sensitivity, and can successfully detect artificially simulated positive horse nose swab samples, which is consistent with the results of the fluorescent PCR detection method, with a compliance rate of 100%. Therefore, the present invention provides a reference idea for the development of multiple detection methods based on RPA-CRISPR / Cas, and has great potential and good application prospects in laboratory and field detection and diagnosis of equine pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0051] Figure 1 It is a schematic diagram of the RPA-CRISPR / Cas13a, 13b, 12a triple detection method of the present invention;

[0052] Figure 2 is an RPA electrophoresis amplification diagram of EAV, AHSV, and HeV in one embodiment of the present invention; wherein A, B, and C represent EAV, AHSV, and HeV, respectively; M: DNA Marker DL2000, 1-7 are 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10, 1copy / μL template, 8 as negative control;

[0053] Figure 3 : is a schematic diagram of the feasibility verification result of the dual CRISPR-Cas detection method in one embodiment of the present invention; wherein AD respectively represent the fluorescence amplification diagrams under four conditions: only the EAV target exists in the system, only the AHSV target exists, both exist at the same time, and no target exists;

[0054] Figure 4 Schematic diagram of the results of condition screening of the dual CRISPR-Cas detection method in one embodiment of the present invention; wherein A is dual CRISPR-Cas reaction buffer screening; B is dual CRISPR-Cas reaction time screening;

[0055] Figure 5 It is a schematic diagram of the results of the specificity verification of the dual CRISPR-Cas detection method in one embodiment of the present invention; wherein A is the fluorescence value result at the end of the reaction; B is the fluorescence visualization result under blue light; HeV-Hendra virus; WNV-West Nile virus.

[0056] Figure 6 : is a schematic diagram of the results of the sensitivity verification of the dual CRISPR-Cas detection method in one embodiment of the present invention; wherein A-1 is the fluorescence change of the FAM channel of the mixed standard positive plasmid with different concentrations; A-2 is the fluorescence change of the ROX channel of the mixed standard positive plasmid with different concentrations; B is the fluorescence value of the mixed standard positive plasmid with different concentrations at the end of the reaction; C is the visualization result under blue light;

[0057] Figure 7 Schematic diagram of the results of the repeatability (n=3) verification of the dual CRISPR-Cas detection method in one embodiment of the present invention; wherein A-1, A-2, and A-3 respectively represent the results of three repeated detections within the group; B-1, B-2, and B-3 respectively represent the results of three repeated detections between the groups;

[0058] Figure 8 Schematic diagram of the feasibility verification result of the triple CRISPR-Cas detection method in one embodiment of the present invention; wherein, EAV: FAM channel; AHSV: ROX channel; HeV: HEX channel;

[0059] Fig. 9 Schematic diagram of the results of fluorescent probe concentration screening in the triple CRISPR-Cas detection method in one embodiment of the present invention; wherein A is the Cas13a-FAM probe concentration screening; B is the Cas13b-ROX probe concentration screening; C is the Cas12a-HEX probe concentration screening;

[0060] Fig.10 Schematic diagram of the results of the specificity verification of the triple CRISPR-Cas detection method in one embodiment of the present invention; wherein WNV-West Nile virus; VEEV-Venezuelan equine encephalitis virus;

[0061] Fig.11: is a schematic diagram of the sensitivity verification result of the triple CRISPR-Cas detection method in one embodiment of the present invention; wherein A is the fluorescence curve graph of FAM, ROX, and HEX channels respectively; B is the fluorescence value at the end of the reaction;

[0062] Fig.12 It is a schematic diagram of the results of the repeatability (n=3) verification of the triple CRISPR-Cas detection method in one embodiment of the present invention; wherein A-1, A-2, and A-3 are respectively the repeatability fluorescence curves within the FAM, ROX, and HEX channel groups, and B-1, B-2, and B-3 are respectively the repeatability fluorescence curves between the FAM, ROX, and HEX channel groups. DETAILED DESCRIPTION

[0063] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0064] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0065] In the following examples, materials and reagents: the live EAV virus and horse nasal swab samples used were stored and provided by Shanghai Customs. The remaining viral plasmids were synthesized by Shanghai Sangon Biotech Co., Ltd. MagaBio plus viral DNA / RNA purification kit was purchased from BioFlux; RPA primers and crRNA sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; ssRNA-RQ was synthesized by GenScript; ssRNA-FQ (RNaseAlert Lab Test Kit V2) was purchased from Invitrogen; ssDNA-HQ was synthesized by Sangon; Cas12a enzyme was purchased from Suzhou Jinan Protein Technology Co., Ltd.; LwaCas13a enzyme was expressed and purified according to the literature method (JS Gootenberg et al., Science, Nucleic acid detection with CRISPR-Cas13a / C2c2, 2017); PsmCas13b enzyme was purchased from SignalChemDiagnostics; RT-RPA detection kit was purchased from TwistDx; HiScribe TM T7 rapid high-yield RNA synthesis kit was purchased from NEB; RNA purification kit was purchased from ZYMO; 5× PrimeScript RT Master Mix and PMD-19T vector were purchased from Takara.

[0066] In the following examples, the instruments used were: XW-80A vortex mixer (Shanghai Qingpu Huxi Instrument Factory); TGear microcentrifuge (Tiangen Biochemical Technology Co., Ltd.); PB602-N electronic balance (Mettle-Toledo); electrophoresis apparatus and electrophoresis tank (Shanghai Tianneng Technology Co., Ltd.); NanoDrop 2000 ultra-micro spectrophotometer and automatic gel imaging system (Thermo Fisher Scientific Inc.); constant temperature amplification instrument (Genie III, OptiGene) (Beijing Shengtaibo Technology Co., Ltd.).

[0067] In the following examples, the template was prepared as follows: RNA was extracted from 300 μL EAV live virus using the MagaBio plus viral DNA / RNA purification kit, and the RNA was incubated at 37°C for 30 min and 85°C for 10 s by reverse transcription into cDNA using 5× PrimeScript RT Master Mix; the cDNA was amplified and connected to the PMD-19T vector to construct the standard plasmid EAV-PMD-19T as a template for subsequent detection of EAV. The AHSV conserved gene VP7 fragment was selected and sent to Shanghai Sangon Biotechnology Co., Ltd. for synthesis, and connected to the pUC57 vector to construct the standard plasmid AHSV-pUC57 as a template for subsequent experimental detection of AHSV. Similarly, the HeV conserved gene fragment was sent to Shanghai Sangon Biotechnology Co., Ltd. for synthesis, and connected to the pUC57 vector to complete the construction of the standard positive plasmid HeV-pUC57. The concentration of the plasmid was measured using a Nanodrop 2000 spectrophotometer and its copy number was calculated. The corresponding volume of enzyme-free water was added to dilute the plasmid to 10 10 The mother solution was diluted tenfold, with a total of 10 dilutions (10 9 ~10 0 The gradient dilution standard positive plasmids were stored in a -20°C refrigerator. The West Nile Virus (WNV) and Venezuelan Equine Encephalitis Virus (VEEV) viral plasmids used for specificity evaluation were stored in the inventor's laboratory.

[0068] The technical solution of the present invention is described exemplarily below.

[0069] Example 1-RPA primer design and crRNA synthesis

[0070] This embodiment designs RPA primers, crRNA, and probes based on the EAV full genome sequence, AHSV VP7 genome sequence, and HeV N gene sequence, specifically including:

[0071] (1) Design of RPA primers;

[0072] The EAV full genome sequence, AHSV VP7 genome sequence, and HeV N gene sequence were downloaded from NCBI for comparison, and the conserved gene fragments were screened out.

[0073] The amplified target sequence information is shown in the following table:

[0074] Table 2 - EAV, AHSV and HeV conserved fragment sequence information

[0075]

[0076] Given that the HeV conserved sequence contains a PAM site suitable for Cas12a detection target design, it was decided to use Cas12a for HeV detection. Primers for EAV, AHSV, and HeV-specific RPA amplification were designed using Primer Premier 5 software, and their sequences are shown in SEQ ID NO.1 to SEQ ID NO.6 in Table 1 above. Due to the characteristics of Cas13 enzyme cleavage of RNA, a T7 RNA polymerase promoter sequence needs to be attached to the 5′ end of the RPA primer to allow T7 transcription.

[0077] (2) Design of crRNA and its transcription template;

[0078] 1) Design of crRNA transcription template: crRNA transcription templates are designed according to the crRNA design principles of Cas13a, Cas13b and Cas12a for the synthesis of crRNA. For EAV, an EAV-crRNA fragment is designed, and its sequence is shown in SEQ ID NO.7 in Table 1 above, and the sequence of its crRNA transcription template is shown in SEQ ID NO.13 to SEQ ID NO.14 in Table 1 above. Since the Cas enzyme detection efficiency is reduced when the crRNA base is mismatched, and the AHSV virus is prone to mutation, and there are multiple single base mutations in its crRNA target sequence, 4 AHSV-crRNA fragments are designed, and their sequences are shown in SEQ ID NO.8 to SEQ ID NO.11 in Table 1 above, and the sequence of its crRNA transcription template is shown in SEQ ID NO.15 to SEQ ID NO.22 in Table 1 above. For HeV, a HeV-crRNA fragment was designed, whose sequence is shown in SEQ ID NO.12 in Table 1 above, and the sequence of its crRNA transcription template is shown in SEQ ID NO.23 to SEQ ID NO.24 in Table 1 above.

[0079] 2) Synthesis of crRNA: Dilute the upstream and downstream sequences of the crRNA transcription template to 10 μM with TE buffer, mix them in equal proportions, take 50 μL of each mixture and put it into the PCR instrument for denaturation at 95°C for 5 minutes, incubate at room temperature for 5 minutes, and anneal to form double-stranded crDNA. For AHSV, it is necessary to mix four double-stranded crDNAs in equal volumes to avoid missed detection due to mismatches, making this method more accurate when detecting actual samples. Use HiScribe TMThe T7 fast and efficient RNA synthesis kit was used to transcribe the crDNA of EAV, AHSV and HeV into crRNA in vitro. The transcription system (total volume 20 μL) was as follows: nuclease-free water, 1.5 μL of 10× reaction buffer, 7.5 mM rNTP, 2 μL of crDNA template, and 2 μL of T7 polymerase. The above system was mixed and incubated at 37°C overnight. The crRNA was purified by an RNA purification kit (ZYMO) and stored at -80°C for later use.

[0080] (3) Probe design;

[0081] According to the specific base cleavage preferences of LwaCas13a protein and PsmCas13b protein, FAM-ssRNA probes were designed for detecting EAV targets, and their sequences are shown in SEQ ID NO.25 in Table 1 above; ROX-ssRNA probes were used to detect AHSV targets, and their sequences are shown in SEQ ID NO.26 in Table 1 above. Based on the characteristics of Cas12a cutting DNA chains, HEX-ssDNA probes were designed to detect HeV targets, and their sequences are shown in SEQ ID NO.27 in Table 1 above. All primers, crRNA transcription templates and probes were synthesized by Shanghai Shenggong Biological Co., Ltd.

[0082] Example 2-Establishment of RPA amplification method

[0083] This example uses standard positive plasmids with different dilutions to perform separate RPA amplification experiments to construct an RPA amplification method, which specifically includes:

[0084] According to the instructions of the RPA amplification kit, 29.5 μL of buffer was thoroughly mixed with 11.2 μL of ddH2O and 2.4 μL of upstream and downstream primers (10 μM) to ensure that the enzyme powder was completely dissolved. Subsequently, 2 μL of cDNA template was added and mixed. Finally, 2.5 μL MgOAc was added. The reactants were mixed and centrifuged and incubated at 37 ° C for 30 minutes. After the RPA product was purified by a DNA purification kit (i.e., a DNA template was obtained), it was detected by 2.0% agarose gel electrophoresis. The gel results were observed with the aid of a chemiluminescence imager. The purified EAV and AHSV products were transcribed in vitro into RNA templates using the T7 fast and efficient RNA synthesis kit for standby use. The upstream and downstream primers corresponding to the above-mentioned viruses are as described in Example 1.

[0085] The above EAV, AHSV and HeV templates were amplified by RPA and verified by agarose gel electrophoresis. The results are as follows Figure 2 As shown in Figure 2, the target sequence bands of EAV, AHSV and HeV were located correctly, and the detection limits were all 10 3 copies / μL.

[0086] Example 3-Establishment and optimization of RPA-CRISPR / Cas13a,13b dual detection method

[0087] This example first verified the feasibility of the RPA-CRISPR / Cas13a, 13b dual detection method, then constructed the RPA-CRISPR / Cas13a, 13b dual detection method for simultaneous detection of EAV and AHSV, and optimized its reaction system buffer and reaction time, specifically including:

[0088] (1) Feasibility of the RPA-CRISPR / Cas13a,13b dual detection method;

[0089] Both LwaCas13a and PsmCas13b are RNA-guided RNases, so they recognize the RNA product transcribed by T7. Since LwaCas13a shows a strong preference for U base cleavage and PsmCas13b has a clear preference for A base cleavage, these unique cleavage preferences are used to design the Cas13a ssRNA reporter gene in the FAM channel and the Cas13bssRNA reporter gene in the ROX channel. The orthogonal reaction triggered by the dual CRISPR-Cas13a / 13b system can detect two targets in the same reaction and distinguish all target combinations. The feasibility verification results are shown in the figure below. Figure 3 As shown in the figure, when the reaction system contains only EAV template, the FAM channel shows an amplification signal, which shows green fluorescence under blue light; when the reaction system contains only AHSV template, the ROX channel shows an amplification signal, which shows red fluorescence under blue light; when both targets exist at the same time, both FAM and ROX channels show amplification signals, which show yellow fluorescence under blue light; when both targets are absent, no probe signal can be detected. The above orthogonal experimental results prove that this method can be used for dual detection of EAV and AHSV.

[0090] (2) Optimization and establishment of CRISPR-Cas13a / 13b dual detection reaction system;

[0091] The RPA amplification method described in Example 2 was used to obtain EAV-RNA templates and AHSV-RNA templates; the CRISPR-Cas13a / 13b reaction system used consisted of 2.5 μL reaction buffer, 2 μL EAV-RNA, 2 μL AHSV-RNA, 1 μM each crRNA, 0.5 μM LwaCas13a, 1 μM PsmCas13b, 100 nM ssRNA-RQ probe, 100 nM ssRNA-FQ probe, and nuclease-free water. The above crRNA and probe are as described in Example 1.

[0092] In this example, nuclease-free water was used as a blank control for the RPA product. The CRISPR-Cas13a / 13b reaction system was placed in a 37°C isothermal amplification system for 10 to 30 minutes, and the FAM and ROX fluorescence intensities were measured every 30 seconds. After the reaction was completed, the eight-tube was placed under a blue light device to take pictures and record the visual results. If the fluorescence value at the end of the reaction exceeded twice the fluorescence value of the negative control, it was judged as positive, otherwise it was negative. Positive samples exhibit yellow fluorescence under blue light (there are two targets).

[0093] 1) Screening of reaction buffer:

[0094] In this example, Cas13 reaction buffer, NEB 1.1, NEB 2.1, NEB 3.1, and T7 reaction buffer were used as buffers of the reaction system for dual CRISPR / Cas detection, and the buffer with the highest detection efficiency was screened out as the reaction buffer of the system.

[0095] Table 3 - Composition of different reaction buffers

[0096]

[0097] The key factor for the success of dual detection is whether the two enzymes are compatible in a single tube reaction, and the reaction buffer greatly affects the reaction compatibility due to its different metal salt ion concentrations and types. Therefore, five different buffers were tested, and the buffer components are shown in Table 3. The reaction results are shown in Table 3. Figure 4 As shown in Part A, NEB1.1, NEB2.1, NEB3.1 and T7 reaction buffer all inhibited the reaction activity of Cas13b, and the ROX channel could not produce fluorescence. At the same time, T7 reaction buffer also inhibited the reaction activity of Cas13a, and the detection efficiency of the FAM channel was low. When the buffer of the reaction system was Cas13reaction buffer, the detection efficiency of both channels was high and the reaction compatibility was good, so Cas13reaction buffer was selected as the buffer of the reaction system.

[0098] 2) After determining Cas13 reaction buffer as the buffer of the reaction system, further optimize the reaction time, add all components into a centrifuge tube and incubate at 37°C for 10min, 15min, 20min, 25min, and 30min, respectively, and analyze the fluorescence intensity to determine the optimal reaction time.

[0099] The above experimental results are as follows Figure 4As shown in part B of the figure, when the dual CRISPR-Cas reaction time was set at 20 min, both the FAM and ROX channels reached their maximum fluorescence values ​​and entered the fluorescence plateau phase, so 20 min was selected as the optimal reaction time.

[0100] Based on the above screening results, an optimal CRISPR-Cas13a / 13b dual detection reaction system (25 μL) was determined: 2.5 μL Cas13 reaction buffer (10×), 2 μL EAV-RNA, 2 μL AHSV-RNA, 1 μM of each crRNA, 0.5 μM LwaCas13a, 1 μM PsmCas13b, 100 nM ssRNA-RQ probe, 100 nM ssRNA-FQ probe and nuclease-free water; the reaction time was: amplification reaction at 37°C for 20 minutes, and the fluorescence intensity was measured every 30 seconds.

[0101] Example 4- Evaluation of the specificity, sensitivity and repeatability of the RPA-CRISPR / Cas13a,13b dual detection method

[0102] This example verifies the specificity, sensitivity and repeatability of the RPA-CRISPR / Cas13a, 13b dual detection system and method constructed in Example 3, specifically including:

[0103] (1) Evaluation of the specificity and sensitivity of the RPA-CRISPR / Cas13a,13b dual detection method;

[0104] In this example, two target virus templates (EAV, AHSV) and other common equine virus nucleic acids (HeV, WNV) were tested by dual CRISPR / Cas detection to evaluate the specificity of the method. 5 The samples were diluted to 1 copy / μL and mixed in equal volumes to serve as templates for the dual CRISPR / Cas reaction system to evaluate the sensitivity of the method. Nuclease-free water was used as a negative control.

[0105] 1) The specificity of the dual CRISPR / Cas approach was evaluated by testing EAV, AHSV, and two other common equine viruses (HeV and WNV). To simulate the dual-target situation, the above four equine viruses were randomly tested in pairs. Figure 5As shown, when the detection templates in the reaction system are EAV and AHSV, both FAM and ROX channels show fluorescence, and yellow fluorescence can be seen under blue light; when the detection template covers any one of EAV or AHSV, only the corresponding fluorescence channel shows fluorescence, and green or red fluorescence can be seen under blue light; when the detection templates are HeV and WNV, neither channel produces fluorescence. Therefore, the dual detection method established by the present invention can accurately identify EAV and AHSV, has no cross-reaction with other equine viruses, and has good specificity.

[0106] 2) EAV and AHSV plasmids were transferred from 10 5 After gradient dilution to 1 copy / μL, equal volumes were mixed and added to the reaction system in sequence as templates. If the fluorescence value at the end of the reaction exceeds twice the maximum fluorescence value of the negative control, it is considered a positive result. Figure 6 As shown, when the mixed plasmid was diluted to 10 2 When the number of copies / μL was 2.0, the FAM and ROX channels could still produce fluorescence, and their fluorescence values ​​were significantly higher than those of the negative control with water as the template. Under blue light, the sample in this tube also showed obvious yellow fluorescence. Therefore, the detection limit of the dual CRISPR / Cas detection method established above was 10 2 The detection limit of the traditional RPA method for EAV and AHSV is 10 3 Compared with the 4000 copies / μL, the dual CRISPR / Cas detection method has an order of magnitude higher sensitivity.

[0107] (2) Evaluation of the repeatability of the RPA-CRISPR / Cas13a,13b dual detection method;

[0108] To evaluate the repeatability of this method, the mixed templates of EAV and AHSV diluted to the same concentration gradient were tested three times within the group, and three times between the groups were tested at the same interval, and a negative control was set up for each test.

[0109] Repeated verification of the same concentration of EAV and AHSV mixed positive plasmids was performed, and the results were as follows Figure 7 As shown, three inter-group experiments and three intra-group experiments were able to successfully detect positive templates with a detection rate of 100%, proving that the method has good repeatability and consistency.

[0110] Example 5-Establishment and optimization of RPA-CRISPR / Cas13a, 13b, 12a triple detection method

[0111] This example first verified the feasibility of the RPA-CRISPR / Cas13a, 13b, 12a triple detection method, then constructed the RPA-CRISPR / Cas13a, 13b, 12a triple detection method for simultaneous detection of EAV, AHSV, and Hev, and optimized its probe concentration, specifically including:

[0112] (1) Feasibility of the RPA-CRISPR / Cas13a, 13b, 12a triple detection method;

[0113] The feasibility verification results of the triple detection method are as follows Figure 8 As shown, when the reaction system contains only EAV, AHSV or HeV templates, only the corresponding single fluorescence channel can collect fluorescence, and other channels cannot detect fluorescence. When the reaction system contains EAV and AHSV targets, FAM and ROX channels produce fluorescence; when the reaction system contains EAV and HeV targets, FAM and HeX channels produce fluorescence; when the reaction system contains AHSV and HeV targets, ROX and HEX channels produce fluorescence. When the three targets are present at the same time, fluorescence can be detected in all three channels, and the negative control does not produce fluorescence, proving that this method can be used for triple detection of EAV, AHSV and HeV.

[0114] (2) Optimization and establishment of the RPA-CRISPR / Cas13a, 13b, 12a triple detection reaction system;

[0115] The RPA amplification method described in Example 2 was used to obtain EAV-RNA template, AHSV-RNA template, and HeV-DNA template; the CRISPR-Cas13a / 13b / 12a reaction system used consisted of 2.5 μL Cas13 reaction buffer (10×), 2 μL EAV-RNA, 2 μL AHSV-RNA, 2 μL HeV-DNA, 1 μM each crRNA, 0.5 μM LwaCas13a, 1 μM PsmCas13b, 0.5 μM LbaCas12a, ROX-ssRNA probe, FAM-ssRNA probe, HEX-ssDNA probe, and nuclease-free water. The above crRNA and probe are as described in Example 1.

[0116] In this example, nuclease-free water was used as the blank control for the RPA product. The system was placed in a 37°C isothermal amplification system for 10 minutes, and the fluorescence intensity was measured every 30 seconds. Regarding the reaction time: From Example 3, it can be seen that the optimal time for the double detection system is 20 minutes, but the triple detection system only needs 10 minutes for the FAM channel to reach the plateau phase, and the positive and negative properties can be well distinguished, so the reaction time of the triple detection system is set to 10 minutes. The fluorescence value at the end of the reaction exceeds twice the fluorescence value of the negative control, which is judged as positive, otherwise it is negative.

[0117] In order to avoid mutual interference and masking of the three fluorescence signals of FAM, ROX and HEX in single-tube detection, which would affect the determination of the positive and negative results, the three probes were screened for concentration. The FAM, ROX and HEX probes were diluted to 200nM, 400nM and 600nM for detection, and the probe concentration with the highest fluorescence value and the closest fluorescence value was selected as the final concentration of the probe in the triple reaction system. Fig. 9 As shown in the figure, the three probe concentrations of FAM, ROX and HEX were screened respectively. When the FAM probe concentration was 400nM, the ROX probe concentration was 600nM, and the HEX probe concentration was 600nM, the three channels could obviously collect fluorescence, and the fluorescence intensity was similar, and there was no situation where the fluorescence signals overlapped each other and were difficult to distinguish. Therefore, the optimal Cas13a-FAM probe concentration of the triple detection system was determined to be 400nM, the Cas13b-ROX probe concentration was 600nM, and the Cas12a-HEX probe concentration was 600nM.

[0118] According to the above screening results, an optimal CRISPR-Cas13a / 13b / 12a triple detection reaction system (25 μL) was determined: 2.5 μL Cas13 reaction buffer (10×), 2 μL EAV-RNA, 2 μL AHSV-RNA, 2 μL HeV-DNA, 1 μM of each crRNA, 0.5 μM LwaCas13a, 1 μM PsmCas13b, 0.5 μM LbaCas12a, 600 nM ROX-ssRNA probe, 400 nM FAM-ssRNA probe, 600 nM HEX-ssDNA probe and nuclease-free water; the reaction time was: amplification reaction at 37°C for 10 min, and the fluorescence intensity was measured every 30 seconds.

[0119] Example 6- Evaluation of the specificity, sensitivity and repeatability of the RPA-CRISPR / Cas13a, 13b, 12a triple detection method

[0120] This example verifies the specificity, sensitivity and repeatability of the RPA-CRISPR / Cas13a, 13b, 12a triple detection system and method constructed in Example 5, specifically including:

[0121] (1) Evaluation of the specificity and sensitivity of the RPA-CRISPR / Cas13a, 13b, 12a triple detection method

[0122] The specificity of the method was evaluated by triple CRISPR / Cas assays against EAV, AHSV, HeV, and two other equine viruses (WNV and VEEV). 5 The copies / μL were gradiently diluted to 1 copy / μL and then mixed in equal volumes, which was used as a template for the triple CRISPR / Cas reaction system to evaluate the sensitivity of the method. Nuclease-free water was used as a negative control.

[0123] 1) If Fig.10 As shown in the figure, EAV, AHSV, HeV and two other equine viruses (WNV, VEEV) were detected. The results showed that when the detection template in the reaction system included EAV, AHSV or HeV, fluorescence appeared in the corresponding channel; when any of WNV and VEEV was introduced as a detection template, no fluorescence appeared and the fluorescence values ​​of other channels were not affected. Therefore, the triple CRISPR-Cas detection method has no cross-reaction with other equine viruses and has good specificity.

[0124] 2) If Fig.11 As shown, EAV, AHSV and HeV templates were respectively 5 The samples were diluted to 1 copy / μL in a gradient manner and mixed in equal proportions for CRISPR triple detection to evaluate its sensitivity. The results showed that the minimum detection concentration of this method for EAV, AHSV and HeV was 10 3 copies / μL.

[0125] (2) Repeatability evaluation of the RPA-CRISPR / Cas13a, 13b, and 12a triple detection method;

[0126] To evaluate the repeatability of this method, the mixed nucleic acids of EAV, AHSV and HeV diluted to the same concentration gradient were tested three times within the group, and three times between the groups were tested at the same interval, and a negative control was set up for each test.

[0127] like Fig.12As shown in the figure, the mixed nucleic acids of EAV, AHSV and HeV diluted to the same concentration gradient were tested three times within the group, and three times between the groups were tested at the same interval, and a negative control was set up for each test. The results showed that both the intra-group and inter-group experiments could detect positive templates, with a detection rate of 100%, proving that the method is repeatable.

[0128] Example 7-RPA-CRISPR / Cas13a, 13b, 12a triple detection method applied to horse nasal swab samples for verification

[0129] This example verifies the practical application of the RPA-CRISPR / Cas13a, 13b, 12a triple detection method constructed in Example 5, specifically including:

[0130] The 30 tubes of horse nasal swab samples stored in the laboratory were pre-tested by CRISPR / Cas and fluorescent RT-PCR methods, and the results were all negative samples, that is, they did not contain EAV, AHSV and HeV viruses.

[0131] In order to evaluate the applicability of the triple CRISPR / Cas detection method in actual samples, 6 horse nose swab samples were randomly selected for artificial simulation of positive samples. Specifically, the samples were mixed with equal volume ratios of EAV, AHSV and HeV plasmids to extract nucleic acid to simulate positive samples; the nucleic acid was extracted by MagaBio plus viral DNA / RNA purification kit for triple CRISPR-Cas detection, and the fluorescence quantitative PCR method was used for verification to evaluate the applicability of the established CRISPR / Cas triple detection method in actual samples. The above experimental results are shown in Table 4 below. The CRISPR / Cas triple detection method can detect positive samples, which is consistent with the results of the fluorescence PCR detection method, with a compliance rate of 100%.

[0132] Table 4 - Triple CRISPR-Cas test results for horse nasal swab samples

[0133]

[0134]

[0135] Note: Tubes 1-6 are simulated positive samples containing EAV, AHSV and HeV; "+" indicates positive, "-" indicates negative

[0136] It can be seen from the above embodiments that the CRISPR-based dual detection method and triple detection method established in the present invention have outstanding advantages in detecting EAV, AHSV and HeV, and have also been verified in artificially simulated positive samples, which fully prepares for the emergence and detection of clinical positive samples. The CRISPR / Cas nucleic acid detection system constructed by the present invention will have greater development potential in the future.

[0137] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. An RPA-CRISPR reaction detection system for detecting equine arteritis virus, African horse sickness virus and / or Hendra virus, characterized in that: The RPA-CRISPR reaction detection system includes an RPA amplification system and a CRISPR-Cas detection system; Wherein, in the RPA amplification system, the primers used to detect equine arteritis virus are shown as SEQ ID NO.1 to SEQ ID NO.2, the primers used to detect African horse sickness virus are shown as SEQ ID NO.3 to SEQ ID NO.4, and the primers used to detect Hendra virus are shown as SEQ ID NO.5 to SEQ ID NO.6; In the CRISPR-Cas detection system, Cas13a is used to detect equine arteritis virus, Cas13b is used to detect African horse sickness virus, and Cas12a is used to detect Hendra virus; the CRISPR-Cas detection system includes crRNA sequences, probes, and RPA amplification products.

2. The RPA-CRISPR reaction detection system according to claim 1, characterized in that: The crRNA sequence for detecting equine arteritis virus is shown in SEQ ID NO.7, and the probe sequence is shown in SEQ ID NO.25; the crRNA sequence for detecting African horse sickness virus is shown in SEQ ID NO.8 to SEQ ID NO.11, and the probe sequence is shown in SEQ ID NO.26; The crRNA sequence for detecting Hendra virus is shown in SEQ ID NO.12, and the probe sequence is shown in SEQ ID NO.27; the 5' end and 3' end of each of the probes are respectively labeled with different fluorescent groups and quenching groups.

3. The RPA-CRISPR reaction detection system according to claim 2, characterized in that: The transcription template sequence of the crRNA sequence for detecting equine arteritis virus is shown as SEQ ID NO.13 to SEQ ID NO.14, the transcription template sequence of the crRNA sequence for detecting African horse sickness virus is shown as SEQ ID NO.15 to SEQ ID NO.22, and the transcription template sequence of the crRNA sequence for detecting Hendra virus is shown as SEQ ID NO.23 to SEQ ID NO.

24.

4. The RPA-CRISPR reaction detection system according to claim 2, characterized in that: The RPA-CRISPR reaction detection system includes an RPA amplification system and a CRISPR-Cas13a / Cas13b dual detection system for detecting equine arteritis virus and African horse sickness virus; Wherein, based on the total volume of the reaction system of 25 μL, the CRISPR-Cas13a / Cas13b dual detection system includes 2.5 μL Cas13 reaction buffer (10×), 2 μL EAV-RNA, 2 μL AHSV-RNA, 1 μM of each crRNA, 0.5 μM LwaCas13a, 1 μM PsmCas13b, 100 nM ssRNA-FQ probe, 100 nM ssRNA-RQ probe and nuclease-free water; wherein, LwaCas13a is used to detect equine arteritis virus, and the ssRNA-FQ probe is a probe for detecting equine arteritis virus; PsmCas13b is used to detect African horse sickness virus, and the ssRNA-RQ probe is a probe for detecting African horse sickness virus.

5. The RPA-CRISPR reaction detection system according to claim 2, characterized in that: The RPA-CRISPR reaction detection system includes an RPA amplification system and a CRISPR-Cas13a / Cas13b / Cas12a triple detection system for detecting equine arteritis virus, African horse sickness virus and Hendra virus; Among them, based on the total volume of the reaction system of 25 μL, the CRISPR-Cas13a / Cas13b / Cas12a triple detection system includes 2.5 μL Cas13 reaction buffer (10×), 2 μL EAV-RNA, 2 μL AHSV-RNA, 2 μL HeV-DNA, 1 μM of each crRNA, 0.5 μM LwaCas13a, 1 μM PsmCas13b, 0.5 μM LbaCas12a, 400 nM ssRNA-FQ probe, 600 nM ssRNA-RQ probe, 600 nM ssDNA-HQ probe and nuclease-free water; wherein, LwaCas13a is used to detect equine arteritis virus, and the ssRNA-FQ probe is a probe for detecting equine arteritis virus; PsmCas13b is used to detect African horse sickness virus, and the ssRNA-RQ probe is a probe for detecting African horse sickness virus; LbaCas12a is used to detect Hendra virus, and the ssDNA-HQ probe is a probe for detecting Hendra virus.

6. The RPA-CRISPR reaction detection system according to claim 4 or 5, characterized in that: The RPA amplification system includes 29.5 μL of reaction buffer, 2.4 μL of each RPA primer, 11.2 μL of ddH2O, 2 μL of cDNA template, and 2.5 μL of MgOAc; wherein the final concentration of each RPA primer in the RPA amplification system is 10 μM.

7. A kit for detecting equine arteritis virus, African horse sickness virus and / or Hendra virus, characterized in that: The kit comprises the RPA-CRISPR reaction detection system as described in any one of claims 1 to 6.

8. A method for detecting equine arteritis virus, African horse sickness virus and / or Hendra virus, characterized in that: Includes steps: S1. After extracting viral nucleic acid from the sample, use the RPA amplification system to amplify each viral cDNA template, and purify the RPA amplification product; wherein the purified equine arteritis virus amplification product and the African horse sickness virus amplification product are transcribed in vitro to obtain their respective RNA templates, and the purified Hendra virus amplification product is a DNA template; S2, using the CRISPR-Cas detection system to identify and cut the target gene in the RNA template and / or DNA template obtained in step S2; S3, determining the detection result based on the fluorescence value or the visual effect under blue light; Wherein, the RPA amplification system and the CRISPR-Cas detection system are as described in any one of claims 1 to 6.

9. The method according to claim 8, characterized in that In step S1, the sample includes a horse nose swab sample, and the reaction conditions of the RPA amplification system are: incubation at 37°C for 30 minutes; In step S2, the reaction conditions of the CRISPR-Cas detection system are: 37° C., isothermal amplification for 10 to 30 minutes, and fluorescence intensity measurement every 30 seconds.

10. A use of the RPA-CRISPR reaction detection system according to any one of claims 1 to 6, or the kit according to claim 7, characterized in that: The application is selected from at least one of the following applications: application in detecting equine arteritis virus, African horse sickness virus and / or Hendra virus for non-diagnostic treatment purposes, and application in screening agents or methods for reducing the concentration of equine arteritis virus, African horse sickness virus and / or Hendra virus.

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